In the description, the following terms are employed:
Analyte--A substance or substances, either alone or in admixtures, whose presence is to be detected and, if desired, quantitated. The analyte may be a DNA or RNA molecule of small or high molecular weight, a molecular complex including those molecules, or a biological system containing nucleic acids, such as a virus, a cell, or group of cells. Among the common analytes are nucleic acids (DNA and RNA) or segments thereof, either single- or double-stranded, viruses, bacteria, cells in culture, and the like. Bacteria, either whole or fragments thereof, including both gram positive and gram negative bacteria, fungi, algae, and other microorganisms are also analytes, as well as animal (e.g., mammalian) and plant cells and tissues.
Probe--A labelled polynucleotide sequence which is complementary to a polynucleotide sequence of a particular analyte and which hybridizes to said analyte polynucleotide sequence.
Label--That moiety attached to a polynucleotide sequence, which as is, which after covalent attachment to it of a signalling moiety or a combination of bridging moiety and signalling moiety or which after non-covalent binding to it of a signalling moiety or a combination of bridging moiety and signalling moiety, gives rise to a signal which is detectable, and in some cases quantifiable. Compounds carrying such labels include, for example, glucosylated nucleotides, glycosylated nucleotides, 5-hydroxymethyluracil, BrdUR, and 5-methylcytosine.
Bridging Moiety--That moiety which on covalent attachment or non-covalent binding to the label of a polynucleotide sequence acts as a link or a bridge between that label and a signalling moiety.
Signalling Moiety--That moiety which on covalent attachment or non-covalent binding to the label of a polynucleotide sequence or to a bridging moiety attached or bound to that label provides a signal for detection of the label.
Signal--That characteristic of a label or signalling moiety that permits it to be detected from sequences that do not carry the signal.
The analysis and detection of minute quantities of substances in biological and non-biological samples has become a routine practice in clinical and analytical laboratories. These detection techniques can be divided into two major classes: (1) those based on ligand-receptor interactions (e.g., immunoassay-based techniques), and (2) those based on nucleic acid hybridization (polynucleotide sequence-based techniques).
Immunoassay-based techniques are characterized by a sequence of steps comprising the non-covalent binding of an antibody and antigen complementary to it. See for example, T. Chard, An Introduction To Radioimmunoassay And Related Techniques (1978).
Polynucleotide sequence-based detection techniques are characterized by a sequence of steps comprising the non-covalent binding of a labelled polynucleotide sequence or probe to a complementary sequence of the analyte under hybridization conditions in accordance with the Watson-Crick base pairing of adenine (A) and thymidine (T), and guanine (G) and cytidine (C), and the detection of that hybridization. [M. Grunstein and D. S. Hogness, "Colony Hybridization: A Method For The Isolation Of Cloned DNAs That Contain A Specific Gene", Proc. Natl. Acad. Sci. USA, 72, pp. 3961-65 (1975)].
In a generalized sense, the non-covalent binding of a labelled sequence or probe to a complementary sequence of an analyte is the primary recognition event of polynucleotide sequence-based detection techniques. This binding event is brought about by a precise molecular alignment and interaction of complementary nucleotides of the probe and analyte. It is energetically favored by the release of non-covalent bonding free energy, e.g., hydrogen bonding, stacking free energy and the like.
In addition to the primary recognition event, it is also necessary to detect when binding takes place between the labelled polynucleotide sequence and the complementary sequence of the analyte. This detection is effected through a signalling step or event. A signalling step or event allows detection in some quantitative or qualitative manner, e.g., a human or instrument detection system, of the occurrence of the primary recognition event.
The primary recognition event and the signalling event of polynucleotide sequence based detection techniques may be coupled either directly or indirectly, proportionately or inversely proportionately. Thus, in such systems as nucleic acid hybridizations with sufficient quantities of radiolabeled probes, the amount of radio-activity is usually directly proportional to the amount of analyte present. Inversely proportional techniques include, for example, competitive immuno-assays, wherein the amount of detected signal decreases with the greater amount of analyte that is present in the sample.
Amplification techniques are also employed for enhancing detection wherein the signalling event is related to the primary recognition event in a ratio greater than 1:1. For example, the signalling component of the assay may be present in a ratio of 10:1 to each recognition component, thereby providing a 10-fold increase in sensitivity.
A wide variety of signalling events may be employed to detect the occurrence of the primary recognition event. The signalling event chosen depends on the particular signal that characterizes the label or signalling moiety of the polynucleotide sequence employed in the primary recognition event. Although the label itself, without further treatment to attach or to bind to it a signalling moiety or a combination of bridging moiety and signalling moiety, may be detectable, it is more usual either to attach covalently or to bind non-covalently to the label a signalling moiety or a combination of bridging moiety and signalling moiety that is itself detectable or that becomes detectable after further modification.
It should, of course, be understood that the combination of bridging moiety and signalling moiety, described above, may be constructed before attachment or binding to the label, or it may be sequentially attached or bound to the label. For example, the bridging moiety may be first bound or attached to the label and then the signalling moiety combined with that bridging moiety. In addition, it should be understood that several bridging moieties and/or signalling moieties may be employed together in any one combination of bridging moiety and signalling moiety.
Examples of the covalent attachment of a signalling moiety or a combination of bridging moiety and signalling moiety to a label are the chemical modification of the label with signalling moieties, such as radioactive moieties, fluorescent moieties or other moieties that themselves provide signals to available detection means or the chemical modification of the label with at least one combination of bridging moiety and signalling moiety to provide that signal.
Examples of the non-covalent binding of a signalling moiety or a combination of bridging moiety and signalling moiety to a label are the non-covalent binding to the label of a signalling moiety that itself can be detected by appropriate means, or the non-covalent binding to the label of a combination of bridging moiety and signalling moiety to provide a signal that may be detected by one of those means. For example, the label of the polynucleotide sequence may be non-covalently bound to an antibody, a fluorescent moiety or another moiety which is detectable by appropriate means. Alternatively, the label could be bound to a bridging moiety, e.g., a lectin, and then bound through the lectin, or bridging moiety, to another moiety that is detectable by appropriate means.
There are a wide variety of signalling moieties and bridging moieties that may be employed for covalent attachment or non-covalent binding to the labels of polynucleotide sequences useful as probes in analyte detection systems. They include both a wide variety of radioactive and non-radioactive signalling moieties and a wide variety of non-radioactive bridging moieties. All that is required is that the signalling moiety provide a signal that may be detected by appropriate means and that the bridging moiety, if any, be characterized by the ability to attach covalently or to bind non-covalently to the label and also the ability to combine with a signalling moiety.
Radioactive signalling moieties and combinations of various bridging moieties and radioactive signalling moieties are characterized by one or more radioisotopes such as .sup.32 P, .sup.131 I, .sup.14 C, .sup.3 H, .sup.60 Co, .sup.59 Ni, .sup.63 Ni and the like. Preferably, the isotope employed emits .beta. or .gamma. radiation and has a long half life. Detection of the radioactive signal is then, most usually, accomplished by means of a radioactivity detector, such as exposure to a film.
Non-radioactive signalling moieties and combinations of bridging moieties and non-radioactive signalling moieties are being increasingly used both in research and clinical settings. Because these signalling and bridging moieties do not involve radioactivity, the techniques and labelled probes using them are safer, cleaner, generally more stable when stored, and consequently cheaper to use. Detection sensitivities of the non-radioactive signalling moieties also are as high or higher than radiolabelling techniques.
Among the preferred non-radioactive signalling moieties or combinations of bridging - signalling moieties useful with non-radioactive labels are those based on the biotin/avidin binding system. [P. R. Langer et al., "Enzymatic Synthesis of Biotin-Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes", Proc. Natl. Acad. Sci. USA, 78, pp. 6633-37 (1981); J. Stavrianopoulos et al., "Glycosylated DNA Probes For Hybridization/Detection of Homologous Sequences", presented at the Third Annual Congress For Recombinant DNA Research (1983); R. H. Singer and D. C. Ward, "Actin Gene Expression Visualized In Chicken Muscle Tissue Culture By Using In Situ Hybridization With A Biotinated Nucleotide Analog", Proc. Natl. Acad. Sci. USA, 79, pp. 7331-35 (1982)]. For a review of non-radioactive signalling and bridging-signalling systems, both biotin/avidin and otherwise, see D. C. Ward et al., "Modified Nucleotides And Methods Of Preparing And Using Same", European patent application No. 63879.
Non-radioactively labelled polynucleotides are not more widely used in detection systems because the attachment of a label, which does not interfere with hybridization, to the polynucleotide sequences that are useful as probes in those detection systems is expensive.
First, the chemical reaction conditions that might be useful for modification of a polynucleotide polymer to add to it a label are often too vigorous to be sufficiently selective for a particular nucleotide. More importantly, chemical labelling of polynucleotide sequences often interferes with hybridization because the label interferes with the hydrogen bonding necessary for hybridization. For example, dicarbonyl reagents, such as kethoxal or glyoxal, react with guanine residues [Shapiro et al., Biochemistry, 5, pp. 2799-2807 (1966); M. Litt, Biochemistry, 8, pp. 3249-53 (1969); Politz et al., Biochemistry, 20, pp. 372-78 (1981)]. However, the kethoxal and glyoxal labelled nucleotides do not hybridize to complementary sequences in the analyte because the label interferes with the hydrogen bonding necessary for hybridization.
Accordingly, in order to label a polynucleotide sequence for use as a probe, a labelled monomeric nucleotide must be synthesized and then incorporated into a polynucleotide sequence. Various methods are available to label an individual nucleotide in such a way that the label does not interfere with hybridization. Various methods, both chemical and enzymatic, are also available to attach those labelled monomeric nucleotides to a polynucleotide probe. For example, a labelled nucleotide, such as 2'-deoxyuridine 5'-triphosphate 5-allylamine biotin may be substituted in DNA probes by nick translation [P. R. Langer et al., "Enzymatic Synthesis Of Biotin-Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes", Proc. Natl. Acad. Sci. USA, 78, pp. 6633-37 (1981)] or by terminal addition to DNA probes using terminal deoxynucleotidyl transferase.
There are, however, production limitations with these processes. For example, it is necessary to synthesize the labelled monomeric nucleotides prior to incorporating them into the polynucleotide probes. This synthesis may sometimes involve expensive chemical processes. The coupling of the labelled monomeric nucleotides into a polynucleotide is also expensive. For example, the enzymes employed in enzymatic coupling are costly. Related limitations include the difficulties and cost in scale-up of such processes to commercial levels. As a result, these processes currently produce non-radioactively labelled polynucleotides that are more costly than are desired.